Disulfide, Sulfide, Sulfoxide, and Sulfone Derivatives of Cyclic Sugars and Uses Thereof

ABSTRACT

In the present invention there are disclosed new derivatives of dianhydrohexite mononitrate corresponding to formula (I), tautomers, pharmaceutically acceptable salts, prodrugs and solvates thereof: 
     
       
         
         
             
             
         
       
     
     as well as pharmaceutical compositions comprising these compounds and uses thereof.

FIELD OF THE INVENTION

The present invention relates to disulfide, sulfide, sulfoxide, andsulfone derivatives of 1,4:3,6-dianhydrohexite mononitrate and their usefor the prevention and/or treatment of vascular disorders.

TECHNICAL BACKGROUND

Nitric oxide (NO) is one of the smallest and simplest of thebiologically active molecules in nature. Moreover, NO appears to be oneof the most ubiquitous molecules in mammalian species. As one of themost widespread signalling molecules, NO is a major player incontrolling nearly every cellular and organ function in the body. NO isthe only endogenous molecule able to function as a neurotransmitter,autacoid, constitutive mediator, inducible mediator, cytoprotectivemolecule, and cytotoxic molecule.

Because NO plays multiple physiological roles in regulating numerous anddiverse organ functions, defects in the NO pathway lead to thedevelopment of many different pathological conditions. These disordersinclude hypertension, atherosclerosis, coronary artery diseases, cardiacfailure, pulmonary hypertension, stroke, impotence, vascularcomplications in diabetes mellitus, gastrointestinal ulcers, asthma, andother central- and systemic-nervous system disorders.

All nitric oxide donors (NODs) share the common property of producingNO-related activity when applied in biological systems and thus mimicendogenous NO responses. However, the pathways leading to NOformation/release differ significantly among the compound classes, as dotheir chemical reactivities. Whereas some compounds require enzymaticcatalysis, others produce NO non-enzymatically. In some compounds, theliberation of NO is preceded by a reduction or an oxidation. The processis complicated still more by the specific susceptibility of compounds tothe changes in pH, oxygen, light and temperature and by the differentby-product formation that takes place during the decomposition or themetabolism. In addition, the kinetics of NO release from a givencompound is often more important than the absolute amount of NOreleased. Moreover, the tissue distribution of the NODs and the sitewhere NO is generated is also of great, importance. All theseconsiderations are important since they explain the very differentpharmacological profiles obtained with the different NODs described inthe literature and make it necessary to fully characterize thepharmacological profile of newly developed NODs in research anddevelopment.

Pharmaceutically useful NODs having a isosorbide-mononitrate skeletonare disclosed in WO 00/20420. The compounds as such disclosed therein donot form part of the present invention. That application describesorganic nitrates capable of providing a potent vasodilating effect andwhich at the same time show a small or null tolerance effect. However,no indication exists for the possible use of said compounds for thetreatment of platelet activation; thrombosis; stroke; tissue damage dueto ischemic and/or to ischemia/reperfusion; pathological conditionswhere oxidative stress plays an important role in their pathogenesis;and/or atherosclerosis. Accordingly the new use of said compounds formspart of the present invention.

One of the principal problems of the nitrated organic compoundsdescribed in the literature and those used clinically resides in thefact that their mechanism of action is the relaxation of vascular smoothmuscle without modifying other pathologic processes involved incardiovascular diseases.

Tissue ischemia results in the depletion of intracellular adenosinetriphosphate (ATP) stores, which subsequently compromises the functionof membrane-associated, ATP-dependent ionic pumps in endothelial cells.This membrane dysfunction allows entry of calcium, sodium, and waterinto the cells. The resultant accumulation of calcium and other ions inthe cell can result in cell swelling and the inappropriate activation ofcellular enzymes. One enzyme that is activated by the rise inintracellular calcium during ischemia is xanthine dehydrogenase (XDH).Under normal conditions, hypoxanthine (a breakdown product of ATPmetabolism) is oxidized by XDH, in an NADPH-dependent manner, to producexanthine and uric acid. However, during the hypoxic condition ofischemia, hypoxanthine levels rise within the cell due to ATPhydrolysis, and there is a calcium-dependent activation of proteasesthat convert the NADPH-reducing XDH to an oxygen-reducing form of theenzyme, namely, xanthine oxidase (XO). On restoration of blood flow(reperfusion) to the tissue and with the reintroduction of molecularoxygen, XO will convert hypoxanthine to xanthine and uric acid, and itwill catalyze the reduction of molecular oxygen to form both superoxideanion radicals (O₂ ⁻) and hydrogen peroxide (H₂O₂). This XO-dependentmechanism of oxygen radical production has been invoked to explain theinvolvement of O₂ ⁻ and H₂O₂ in reperfusion injury to a variety oforgans, including intestine, brain, heart, and skeletal muscle.

The generation of oxygen radicals in postischemic tissues appears toovercome the capacity of endogenous antioxidants such as superoxidedismutase (SOD), catalase, glutathione to protect endothelial andparenchymal cells, exogenous antioxidants such as SOD and catalase havebeen shown to attenuate the leukocyte infiltration and tissue injuryelicited by ischemic and reperfusion.

Nitric oxide bioavailability appears to be reduced in reperfusion, whichis likely due to a decline in endothelial NO production and an increasedinactivation of NO by endothelial-cell-derived O₂ ⁻. The limitedbioavailability of NO contribute to the abnormal cell-cell interactionsand vascular dysfunction during reperfusion. Nitric oxide-donatingcompounds have shown promise as protective agents in experimental modelsof ischemia-reperfusion. However, considering the processes involved inthe damage by ischemia-reperfusion it would be of great interest to havea molecule, with both properties: being a NO-donor and at the same timewith antioxidant properties.

Atherosclerosis is an active process initiated by a continuous damage ofthe vascular endothelium. The view of atherosclerosis as a response to adamage of the endothelium was developed when the association ofatherosclerosis with risk factors (high LDL plasma levels, low HDLplasma levels, hypertension, oxidative stress, tobacco consumption,diabetes mellitus, high Lp(a) plasma levels or modification of LDL suchas oxidation or glycation that prevent LDL removal by the specificreceptors) was studied. LDL accumulates in the vascular wall as aconsequence of a vascular endothelial cells active transport. Duringthis process, the LDL suffers the oxidation of a part of the molecule.The presence of oxidated-LDL (ox-LDL) is of capital importance in thedevelopment of the atherosclerotic lesion. To the same extent, thetheory that oxidized LDL is responsible for some of the pathologicalfeatures of atherosclerotic lesions derives from the findings incultured cells systems that oxidized LDL causes cellular changes thatcorrelate with known aspects of arterial lesions but are not induced bynative LDL. Endothelial injury, LDL retention in intimal interstitium,monocyte recruitment into intima, engorgement of macrophages withlipoprotein-derived lipid, smooth muscle cell migration andproliferation, accumulation of necrotic cell debris, and tendenciestowards vasoconstriction and procoagulant activity are characteristicsof atherosclerosis.

Cardiac allograft vasculopathy is an unusually accelerated and diffuseform of coronary atherosclerosis that limits the long-term success ofcardiac transplantation. Coronary endothelial vasodilator dysfunction isa common and early marker for the development of cardiac allograftvasculopathy.

Accordingly, novel nitrated organic compounds which, in addition to thevasodilating activity, could combine activities that would allow them tomodify other pathologic processes involved in cardiovascular diseases,such as atherosclerosis aid tissue damage due to ischemia and/or due toischemia and reperfusion, will represent an important advantage withrespect to the compounds nowadays in use.

SUMMARY OF THE INVENTION

An object of the invention is a novel type of compounds, derivatives ofdianhydrohexite mononitrate, which are capable of providing a potentvasodilating effect and which at the same time modify other pathologicprocesses involved in cardiovascular diseases such as, atherosclerosis,cardiac allograft vasculopathy, and tissue damage due to ischemia and/orto ischemia-reperfusion.

Another object of the invention is a novel type of compounds,derivatives of dianhydrohexite mononitrate, which are capable ofproviding a potent antithrombotic effect even at a dose that does notmodify the blood pressure.

Another object of the invention is a novel type of compounds,derivatives of dianhydrohexite mononitrate, which are capable ofproviding a synergistic effect with thrombolytic drugs, anticoagulants,antithrombotics, antioxidants and hypolipemiant drugs.

A further object of the present invention relates to the new use ofderivatives of dianhydrohexite mononitrate for the manufacture of apharmaceutical composition for the treatment of cardiovascular disordersrelated to atherosclerosis.

DETAILED DESCRIPTION OF THE INVENTION

It is an object of the present invention to provide a compound accordingto formula (I), or a tautomer, a pharmaceutically acceptable salt, aprodrug or a solvate thereof:

wherein:n is an integer of 0, 1, or 2,X represents —S(O)_(m)—, —(C═O)— or a single bond, wherein m is aninteger of 0, 1, or 2; with the proviso that when X represents —(C═O)—then n is 0,R represents hydrogen or is a residue R^(a), which residue R^(a) isselected from the group consisting of:

-   -   C₁₋₆ alkyl;    -   C₂₋₆ alkenyl;    -   C₃₋₈ cycloalkyl;    -   C₃₋₈ cycloalkyl, wherein one CH₂ group is replaced by O,    -   S, NH or NCH₃;    -   C₄₋₈ cycloalkenyl;    -   C₄₋₈ cycloalkenyl, wherein one CH₂ group is replaced by O, S, NH        or NCH₃;    -   phenyl;    -   pyridyl;    -   thiophenyl;    -   nitrosyl;    -   S-cysteinyl;    -   S-glutathionyl; and

-   -   -   wherein R* is selected from the group consisting of            hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₃₋₈ cycloalkyl, C₄₋₈            cycloalkenyl, acetyloxy, hydroxyl, ONO₂ and halogen,

    -   wherein R^(a) optionally is substituted by one to three groups        independently selected from C₁₋₆ alkyl, C₂₋₆ alkenyl, C₃₋₈        cycloalkyl, C₄₋₈ cycloalkenyl, acetyloxy, hydroxyl, ONO₂ and        halogen.

It is preferred in the compounds according to formula (I) whenRXS(O)_(n)— and —ONO₂ are trans to each other with respect to the ringplane, as depicted in formulae (Ia) and (Ib):

that then RXS(O)_(n)— does not represent

wherein Z is an C₁-C₄ alkyl group, aryl group, or an aralkyl group.

Surprisingly, in addition to the potent vasodilating effect with smallor null tolerance, the compounds of formula (I) possess anti plateletactivation, anti thrombotic, anti stroke, anti oxidant, anti tissueinjury/damage due to ischemia and/or ischemia/reperfusion, and antiatherosclerotic properties.

Another embodiment of the present invention relates to a pharmaceuticalcomposition comprising as active ingredient at least one of thederivatives of dianhydrohexite mononitrate according to formula (I), atautomer, a pharmaceutically acceptable salt, a prodrug or a solvatethereof.

A further embodiment of the present invention relates to the use of atleast one derivative of dianhydrohexite mononitrate according to formula(I), a tautomer, a pharmaceutically acceptable salt, a prodrug or asolvate thereof as active ingredient for the manufacture of apharmaceutical composition for the prevention and/or treatment ofatherosclerosis, endothelial dysfunctions, vasospasm, cardiac allograftvasculopathy, dysfunctions of the circulatory system, plateletactivation, thrombosis, stroke, pathological conditions where oxidativestress plays an important role in their pathogenesis such as but notlimited to Alzheimer's disease, pathological conditions where a deficitof nitric oxide plays an important role in their pathogenesis, and/ortissue damage due to ischemia and/or due to ischemia-reperfusion.

The above used expressions will be outlined in more detail below:

The expression “pharmaceutically acceptable salt, solvate or prodrugthereof” describes any pharmaceutically acceptable salt, eater, solvateor any other compound that, administered to a patient (directly orindirectly), provides a compound described herein. Nevertheless, it willbe considered that the pharmaceutically non acceptable salts also areincluded within the limits of this invention since these compounds canbe useful in the preparation of pharmaceutically acceptable salts.Preparation of salts, prodrugs and derivatives can be carried out bymethods known in the state of the art.

For example, pharmaceutically acceptable salts of compounds describedherein are synthesized from the corresponding compound, that contains anacid or basic group, by conventional chemical methods. Generally, thesesalts are, for example, prepared by means of the reaction of free acidicor basic forms of these compounds in a stoichiometric amount with acorresponding base or acid in water or an organic dissolvent or amixture of both. Non-aqueous media like ether, ethyl acetate,isopropanol or acetonitrile are generally preferred. Examples of acidsalts include mineral acid salts such as hydrochloride, hydrobromide,hydriodide, sulphate, nitrate, phosphate and organic acid salts such asacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate,mandelate, methylsulphonate and p-toluensulphonate.

Examples of basic salts include inorganic salts such as salts of sodium,potassium, calcium, ammonium, magnesium, aluminium and lithium, andorganic salts such as ethylenediamine, ethanolamine,N,N-dialkyleneethanolamine, triethanolamine, glucamine and basic saltsof amino acids.

The particularly preferred derivatives or prodrugs are those thatincrease the bioavailability of compounds of the present invention whensuch compounds are administered to a patient (for example, allowing thatan administered compound of oral form more quickly is absorbed in blood)or those that increase the liberation of the corresponding compound to abiological compartment (for example, the brain or the lymphatic system).

Any compound that is a prodrug of a compound of formula (I), belongs tothe scope of the invention. The term “prodrug” is used in the amplestsense and includes derivatives that “in vivo” are metabolized intocompounds of the invention. Such derivatives include, depending on thepresent functional groups in the molecule and without limitation, thefollowing derivatives: esters, esters of amino acids, phosphate esters,metallic salts of sulfonated compounds, carbamates and amide, esters.

The compounds of the present invention can preferably be in theircrystalline form, or like free compounds or solvates. The solvationmethods which can be applied are those generally known in the art. Thesuitable solvates are pharmaceutically acceptable solvates. It ispreferred that the solvate is a hydrate.

It is preferred that the compounds of formula (I), or their salts orsolvates are in their acceptable pharmaceutically substantially pureform. By pharmaceutically acceptable form it is understood, “interalia”, having an pharmaceutically acceptable level of purity excludingusual pharmaceutical additives such as diluents and carriers, andincluding material considered non-toxic at levels of normal doses. Thelevels of purity for the drug are over 50%, preferably over 70% andstill more preferable over 90%. In an even more preferred embodiment thecompounds of formula (I), or the salts, solvates or prodrugs thereofhave purity over 95%.

The compounds of the present invention represented by the formula (I)can include enantiomers, depending on the presence of chiral or isomericcenters, and/or depending on the presence of multiple bonds (forexample, Z, E). The pure isomers, enantiomers or diastereoisomers andtheir mixtures are within the scope of the present invention.

The term “halogen” refers to fluorine, chlorine, bromine, or iodine,whereof bromine is preferred.

The term “C₁₋₆ alkyl” as used herein refers to a straight or branchedchain alkyl moiety having from 1 to 6 carbon atoms, including forexample, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl,sec-butyl, t-butyl, pentyl, and hexyl.

The term “C₂₋₆ alkenyl” as used herein refers to a straight or branchedchain alkenyl moiety having from 2 to 6 carbon atoms and at least onedouble bond of either E or Z stereochemistry where applicable. This termwould include, for example, vinyl, allyl, 1- and 2-butenyl, and2-methyl-2-propenyl.

The term “C₃₋₈ cycloalkyl” as used herein refers to an alicyclic grouphaving from 3 to 8 carbon atoms. Some illustrative examples of suchcycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl andcyclohexyl.

Accordingly, the term “C₃₋₈ cycloalkyl wherein one CH₂ group is replacedby O, S, NH or NCH₃” refers to an alicyclic group having from 3 to 8carbon atoms wherein one CH₂ group is replaced by O, S, NH or NCH₃. Someillustrative examples of such groups are tetrahydropyrane,tetrahydrofurane, pyrrolidine, piperidine, and tetrahydrothiophene.

The term “C₄₋₈ cycloalkenyl” refers to an alicyclic group having from 4to 8 carbon atoms. Some illustrative examples of such cycloalkenylgroups are cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenyl.

It is preferred that R represents hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl,C₃₋₈ cycloalkyl, C₄₋₈ cycloalkenyl, (C₁₋₆ alkyl)C₃₋₈ cycloalkyl, (C₁₋₆alkyl)C₄₋₈ cycloalkenyl, phenyl or (C₁₋₆ alkyl)phenyl, whereas C₁₋₆alkyl is especially preferred.

It is further preferred that in formula (I) either one or both of m andn is 0.

Also it is preferred that X represents a single bond or —S—.

It is especially preferred that the compounds of formula (I) correspondto compounds which are represented by formula (Ia) and/or formula (Ib):

The compounds of formula (I) also include (R) and (S) diastereoisomersaccording to the formulas (Ic) and (Id):

Especially preferred compounds of formula (I) area

-   2-thioisosorbide 5-mononitrate,-   5,5′-dinitrato-2,2″-dithiodiisosorbide,-   2-methylthioisosorbide 5-mononitrate,-   2-[(R)-methylsulfinyl]isosorbide 5-mononitrate,-   2-[(S)-methylsulfinyl]isosorbide 5-mononitrate,-   2-methyl-sulfinylisosorbide 5-mononitrate,-   2-methylsulfonylisosorbide 5-mononitrate,-   S-nitroso-2-thioisosorbide 5-mononitrate,-   2-(tetrahydropyran-2-yl-thio)isosorbide 5-mononitrate,-   2-(isosorbidyl-2′-dithio)isosorbide 5-mononitrate, and-   2-(5′-acetyloxyisosorbidyl-2′-dithio)isosorbide 5-mononitrate.

Further, it is especially preferred to use2-acetylthio-isosorbide-5-mononitrate (compound (12)), a tautomer, apharmaceutically acceptable salt, a prodrug and/or a solvate thereof:

as active ingredient for the manufacture of a pharmaceutical compositionfor the prevention and/or treatment of thrombosis, ischemia, cell/tissuedamage induced by ischemia and/or by ischemia and reperfusion,hypertension, vasospasm, atherosclerosis and/or cardiac graftvasculopathy.

Additionally, the compounds of formula (I), especially2-acetylthioisosorbide 5-mononitrate (12), their tautomers,pharmaceutically acceptable salts, prodrugs and solvates thereof, mayalso be used in a therapy for the prevention and/or treatment ofatherosclerosis, cardiac allograft vasculopathy, platelet activation,thrombosis, stroke, pathological conditions where oxidative stress playsan important role in their pathogenesis, and/or tissue damage due toischemia and/or due to ischemia-reperfusion. These compounds mayespecially be used in the prevention and/or treatment of pathologicalconditions, where oxidative stress plays an important role in theirpathogenesis, such as allergy, stroke, Alzheimer's disease, and/orischemic cardiovascular diseases. The pharmaceutical compositions may beadministered by different routes. For example, they may be administeredorally in form of pharmaceutically preparations such as tablets,capsules, syrups and suspensions. Parenterally in form of solutions oremulsions, etc. They may also be administered topically in form ofcreams, pomades, balsams, etc., and transdermically for example throughthe use of patches or bandages. They may also be applied directly in therectum as suppositories. The preparations may comprise physiologicallyacceptable carriers, excipients, activators, chelating agents,stabilizers, etc. In case of injections there may be incorporatedphysiologically acceptable buffers, solubilizing agents or isotonics.

The pharmaceutical compositions according to the present invention mayfurther comprise a thrombolytic agent, preferably plasminogen activatorurokinase, streptokinase, alteplase or anistreplase. They may alsocontain an anticoagulant agent, preferably heparin, dicoumarol,acenocoumarol, enoxaparine or pentosan polysulfate. Moreover, they maycontain additionally an antithrombotic agent preferably acetyl salicylicacid, dipyridamole, ticlopidine, clopidrogel, triflusal, pentosanpolysulfate or abciximab. They can further comprise an immunoglobulin orfragment thereof having A specificity for, glycoprotein IIb/IIIa.

Alternately, the pharmaceutical compositions according to the inventionmay further comprise an hypolipemiant agent preferably simvastatin,lovastatin, atorvastatin, pravastatin, fluvastatin, eptastatin,lifibrol, acifran, acitemate, glunicate or rosuvastatine. They may alsocontain an antioxidant/free radical scavengers agent, preferablyselected from nicaraven, ranolazine, emoxipin, glutatione, edaravone,raxofelast, lycopene, N-acetyl-L-cysteine, N-acetyl-D-cysteine, aracemic mixture of N-acetyl-L-cysteine and N-acetyl-D-cysteine, orcarvedilol.

The pharmaceutical compositions according to the present invention maybe used for the treatment and/or prevention of atherosclerosis, cardiacallograft vasculopathy, platelet activation, thrombosis, stroke, tissuedamage due to ischemia and/or due to ischemia-reperfusion, and/orpathological conditions where oxidative stress plays an important rolein their pathogenesis (such as but not limited to allergy, stroke,Alzheimer's disease, ischemic cardiovascular diseases); and/orpathological conditions where a deficit of NO plays an important role intheir pathogenesis. They can also be used for the treatment and/orprevention of dysfunctions of the circulatory system preferablycardiovascular and coronary dysfunctions.

The daily dose may be varied depending on the specific symptoms, theage, the body weight of the patients, the specific mode ofadministration, etc., and a daily normal dose for an adult person couldbe between 0.1 to 500 mg, and could be administered as one dose only ordivided into several doses during the day.

The compounds of the present invention can be prepared by preparationmethods known in the art, by adaptation of the known processes by theskilled person or by a new process described below.

Hence, another embodiment of the present invention relates to aprocesses for preparing compounds of formula (I), tautomers,pharmaceutically acceptable salts, prodrugs and solvates thereof.

According to the invention it is preferable to prepare a compound of theformula (I), a tautomer, a pharmaceutically acceptable salt, a prodrugor a solvate thereof by the process outlined below:

wherein;n is an integer of 0, 1, or 2,X represents —S(O)_(m)— or a single bond, wherein m is an integer of 0,1, or 2,and R represents hydrogen or is a residue R^(a), which residue R^(a) isselected from the group consisting of:

-   -   C₁₋₆ alkyl;    -   C₂₋₆ alkenyl;    -   C₃₋₈ cycloalkyl;    -   C₃₋₈ cycloalkyl, wherein one CH₂ group is replaced by O, S, NH        or NCH₃;    -   C₄₋₈ cycloalkenyl;    -   C₄₋₈ cycloalkenyl, wherein one CH₂ group is replaced by O, S, NH        or NCH₃;    -   phenyl;    -   pyridyl;    -   thiophenyl;    -   nitrosyl;    -   S-cysteinyl;    -   S-glutathionyl; and

-   -   wherein R* is selected from the group consisting of hydrogen,        C₁₋₆ alkyl, C₂₋₆ alkenyl, C₃₋₈ cycloalkyl, C₄₋₈ cycloalkenyl,        acetyloxy, hydroxyl, ONO₂ and halogen,    -   wherein R^(a) optionally is substituted by one to three groups        independently selected from the group consisting of C₁₋₆ alkyl,        C₂₋₆ alkenyl, C₃₋₈ cycloalkyl, C₄₋₈ cycloalkenyl, acetyloxy,        hydroxyl, ONO₂ and halogen,        which process comprises conducting the following steps:    -   a) effecting the hydrolysis of a compound of formula (IIa):

-   -   -   wherein R′ is C₁-C₆ alkyl, preferably methyl, to obtain the            following compound:

-   -   -   and

    -   (b) optionally, effecting on the compound prepared according to        the step (a):        -   I. an oxidation reaction to obtain:

-   -   -   -   optionally followed by a second oxidation to obtain the                following compound:

-   -   -   -   wherein:            -   n is 1 or 2,            -   X is —S(O)_(m)—, wherein m is 0, 1 or 2, and            -   R* represents hydroxyl or ONO₂;

        -   II. a substitution reaction to obtain:            -   a compound according to formula (I), wherein:            -   n is an integer of 0,            -   X represents a bond,            -   and R does not represent nitrosyl,            -   optionally followed by an oxidation to obtain a compound                according to formula (I), wherein:            -   n is an integer of 0,            -   X represents —S(O)_(m)—, wherein m is an integer of 0 or                1,            -   and R does not represent nitrosyl;

        -   III. a substitution reaction to obtain:            -   a compound according to formula (I), wherein:            -   n is an integer of 0, and            -   X represents —S—;            -   optionally followed by an oxidation to obtain a compound                according to formula (I), wherein:            -   n is an integer of 1 or 2, and            -   X represents —S(O)_(m)—, wherein m is 0, 1 or 2; or

        -   IV. a nitrosation reaction to obtain:

According to the above process of the invention it is especiallypreferable to prepare a compound of the formula (Ia), a tautomer, apharmaceutically acceptable salt, a prodrug or a solvate thereof:

wherein n, X, m and R have the above meaning,and wherein said process comprises conducting the following steps:

-   -   a) effecting the hydrolysis of a compound of formula (II):

-   -   -   wherein R′ is C₁-C₆ alkyl, preferably methyl, to obtain            2-thioisosorbide 5-mononitrate (1),

-   -   -   and

    -   (b) optionally, effecting on compound (1) prepared according to        the step (a):        -   I. an oxidation reaction to obtain:            -   5,5′-dinitrato-2,2′-dithiodiisosorbide (2) or            -   2-(isosorbidyl-2′-dithio)isosorbide            -   5-mononitrate (8),            -   optionally followed by a second oxidation to obtain a                compound according to formula (Ie):

-   -   -   -   wherein:            -   n is 1 or 2,            -   X is —S(O)_(m)—, wherein m is 0, 1 or 2, and            -   R* represents hydroxyl or ONO₂,

        -   II. a substitution reaction to obtain:            -   a compound according to formula (Ia), wherein:            -   n is an integer of 0,            -   X represents a bond,            -   and R does not represent nitrosyl,            -   optionally followed by an oxidation to obtain a compound                according to formula (Ia), wherein:            -   n is an integer of 0,            -   X represents —S(O)_(m)—, wherein m is an integer of 0 or                1,            -   and R does not represent nitrosyl;

        -   III. a substitution reaction to obtain;            -   a compound according to formula (Ia), wherein:            -   n is an integer of 0, and            -   X represents —S—;            -   optionally followed by an oxidation to obtain a compound                according to formula (Ia), wherein:            -   n is an integer of 1 or 2, and            -   X represents —S(O)_(m)—, wherein m is 0, 1 or 2; or

        -   IV. a nitrosation reaction to obtain:            -   S-nitroso-2-thioisosorbide 5-mononitrate (6).

Optional step (b) of this new process of the invention is describedschematically in Schemes 1 to 4 for specific compounds of the invention.These Schemes 1 to 4 relate to the oxidation reaction I, thesubstitution reaction II, the substitution reaction III, and thenitrosation reaction IV, respectively.

A specifically preferred process of the invention includes steps (a) and(b) II for the preparation of:

-   2-[(R)-alkylsulfinyl]isosorbide 5-mononitrate and/or-   2-[(S)-alkylsulfinyl]isosorbide 5-mononitrate.

It is further preferred that both diastereoisomers are separatedsubsequently, which separation may be carried out by using conventionalmethods known in the art.

A further preferred preparation process of the invention concerns thepreparation of a compound of formula (II) or a tautomer, apharmaceutically acceptable salt, a prodrug or a solvate thereof:

which process comprises the following steps:

-   -   a) effecting an oxidation reaction of a compound of formula        (III):

-   -   wherein R′ is C₁-C₆ alkyl, preferably methyl, to obtain        2,2′-dithio-diisosorbide (10),

-   -   -   and

    -   (b) effecting a nitration reaction of the compound prepared in        step (a) with a nitrating agent in the presence of a carboxylic        anhydride, preferably acetic anhydride.

Finally, another embodiment of the present invention relates to2,2′-dithiodiisosorbide, compound (10), which is an intermediatecompound in the preparation of compound (11) of the invention.

In the working examples herein (vide infra) there are described indetail suitable processes to obtain various of the compounds accordingto the general formula (I). In view of these examples, it is within theskilled persons general knowledge to obtain the compounds not explicitlyexemplified herein via suitable modifications of the working examplesherein. It will be apparent to the skilled person that these examplesare solely for illustrative purposes and must not be considered to limitthe invention.

EXAMPLES

The compounds obtained in the examples that appear below are identifiedby their proton (¹H-NMR) and carbon-13 (¹³C-NMR) nuclear magneticresonance spectroscopy data.

The nuclear magnetic resonance spectra were recorded with VarianGemini-2000 or Varian Gemini-300 spectrometers.

The operating frequency and the solvent used to record the spectrum areindicated in the ¹H-NMR spectra. The position of the signals isindicated in δ (ppm), with the signal of the solvent protons taken asthe reference. The reference values were 7.24 ppm for deuteratedchloroform and 2.49 ppm for deuterated dimethyl sulfoxide. The signalobtained from tetramethylsilane (TMS) protons is occasionally taken asan internal reference, with 0 ppm used as a reference value. The numberof protons for each signal as measured by electronic integration and thetype of signal are indicated in parentheses, using the followingabbreviations: s (singlet), d (doublet), t (triplet), dd (doublet ofdoublets), ddd (doublet of doublet of doublets), bs (broad signal), cs(complex signal), s.a. D₂O (simplifies upon deuteration), d.a. D₂O(disappears upon deuteration).

The operating frequency and the solvent used in each spectrum areindicated in the ¹³C-NMR spectra. The position of the signals isindicated in δ (ppm), with the signal of the solvent carbons taken asthe reference. The reference values are 77.00 ppm for deuteratedchloroform and 39.50 ppm for hexadeuterated dimethyl sulfoxide.

In some cases, nuclear magnetic resonance experiments were also carriedout using the pulse sequences APT (Attached Proton Test), HETCOR(Heteronuclear Chemical Shift Correlation) or COSY (CorrelatedSpectroscopy) as an aid to assignment.

In the experimental part the following abbreviations are used:

-   -   AcOEt ethyl acetate    -   AcOH acetic acid    -   DMSO-d₆ hexadeuterated dimethyl sulfoxide    -   EtOH ethyl alcohol    -   EtOEt ethyl ether    -   HPLC high performance liquid chromatography    -   Hx hexane    -   MeI methyl iodide    -   MeOH methyl alcohol    -   LC-(ApcI)MS liquid chromatography-atmospheric pressure chemical        ionization mass spectrometry    -   s.d. standard deviation    -   s.e.m. standard error of mean    -   THP tetrahydropyranyl

Example 1 Method to obtain 2-thioisosorbide 5-mononitrate (1)

In a 50 mL flask, 1.00 g (4.02 mmol) of 2-acetylthioisosorbide5-mononitrate (12) obtained according to WO 00/20420 were dissolved in20.0 mL of methyl alcohol. 10.0 mL of a 10% methanol solution of sodiumhydroxide were added all at once. After rapidly covering and stirringfor 1 min at room temperature (ca. 25° C.), 2.23 mL of concentratedhydrochloric acid were added all at once. It was stirred andconcentrated until dryness, eliminating the solvent at reduced pressureat a temperature below 30° C.

The residue was suspended in chloroform. This chloroform solution wasfiltered and then dried over anhydrous magnesium sulfate. Afterfiltering the solvent was eliminated at reduced pressure. It was driedat reduced pressure to obtain 0.83 g of an orange-yellow oilcorresponding to the product of interest. Yield: 100%.

¹H-NMR (200 MHz, CDCl₃): 5.36-5.26 (1H, m, CHONO₂), 4.95 (1H, t, J=5.0Hz, CHCHONO₂), 4.42 (1H, d, 4.8 Hz, CHCHS), 4.07 (1H, dd, J=4.6 Hz,J=4.4 Hz, H—CHCHS), 3.97 (1H, dd, Hz, J=2.5 Hz, H—CHCHONO₂), 3.87-3.76(2H, cs, H—CHCHS, H—CHCHONO₂), 3.45-3.35 (1H, m, CHS), 1.77 (1H, d,J=8.6 Hz, SH).

¹³C-NMR (50 MHz, CDCl₃): 91.21 (CHCHS), 81.22 (CHONO₂), 81.07(CHCHONO₂), 76.15 (CH₂CHS), 69.26 (CH₂CHONO₂), 42.82 (CHS).

Example 2 Method to obtain 5,5′-dinitrato-2,2′-dithiodiisosorbide (2)

Procedure 1:

0.50 g of 2-acetylthioisosorbide 5-mononitrate (12) obtained accordingto WO 00/20420 was dissolved in 10.0 mL of methyl alcohol. This solutionwas slowly added, drop by drop, to 200 mL of human plasma in a 250 mLflask with strong magnetic stirring. The reaction mixture was stirred atroom temperature for 15 hours. The reaction crude was poured over 500.0mL of acetonitrile while stirring vigorously, observing the instantprecipitation of a flocculant white solid corresponding to the plasmaproteins. It was centrifuged at 3000 rpm and at 20° C. for 30 min, theliquid separated and the solid (protein mass) suspended over 250.0 mL ofacetonitrile. It was stirred and centrifuged under the same conditionsas above (3000 rpm/20° C./30 min).

The supernatant liquor was decanted and combined with the previous one.The solvent was evaporated at reduced pressure at a temperature below30° C. The resulting aqueous residue (about 200 mL) was extracted with4×500 mL chloroform. The organic phases were combined and dried overanhydrous magnesium sulfate. After filtering the solvent wasconcentrated at reduced pressure. This results in 350 mg of a whitesolid which is purified by chromatography: (CHCl₃/AcOEt 6:1), isolating250 mg of a white solid corresponding to the 2-thioisosorbide5-mononitrate disulfide product (2). Yield: 60%.

Procedure 2:

In a 50 mL flask, 1.00 g (4.02 mmol) of 2-acetylthioisosorbide5-mononitrate (12) obtained according to WO 00/20420 was dissolved in 20mL of methyl alcohol and 10 mL of a 10% methanol solution of potassiumhydroxide were added. The reaction mixture was covered and stirred for 5hours at room temperature. The precipitation of a white solidcorresponding to the disulfide (2) is observed during the reaction. Thesolid was filtered off and washed several times with methyl alcohol.Drying at reduced pressure, yields 0.58 g of a white solid correspondingto the 2-thioisosorbide 5-mononitrate disulfide product of interest (2).Yield: 70%.

¹H-NMR (200 MHz, DMSO-d₆): 5.51-5.43 (2H, m, 2CHONO₂), 4.95 (2H, t,J=5.4 Hz, 2CHCHONO₂), 4.51 (2H, d, J=4.8 Hz, 2CHCHS), 4.04-3.73 (10H,cs, 2CH₂CHONO₂, 2CH₂CHS, 2CHS).

¹³C-NMR (50 MHz, DMSO-d₆): 88.46 (2CHCHS), 83.31 (2CHONO₂), 81.50(2CHCHONO₂), 73.24 (2 CH₂CHS), 69.95 (2CH₂CHONO₂), 54.01 (2CHS).

Example 3 Method to obtain 2-methylthioisosorbide 5-mononitrate (3)

In a 50 ml, flask, 1.00 g (4.02 mmol) of 2-acetylthioisosorbide5-mononitrate (12) obtained according to WO 00/20420 were dissolved in20.0 mL of methyl alcohol and 5.0 mL of a 10% methanol solution ofpotassium hydroxide was added all at once. The reaction mixture wascovered and stirred for 5 minutes at room temperature. 2.0 mL of methyliodide (32.00 mmol) were added all at once, the mixture covered andstirred for 2 hours at room temperature. It was concentrated to dryness,eliminating the solvent at reduced pressure. The residue was dissolvedin 250 mL of chloroform and washed with 50 mL of water. The organicphase was separated and washed with 3×50.0 mL of water.

After drying over anhydrous magnesium sulfate, it was filtered and thesolvent eliminated at reduced pressure. After drying at reducedpressure, this results in 0.68 g of a white solid corresponding to theproduct of interest. Yield: 76%.

¹H-NMR (200 MHz, CDCl₃): 5.34-5.26 (1H, m, CHONO₂), 4.93 (1H, t, J=5.2Hz, CHCHONO₂), 4.48 (1H, d, J=4.8 Hz, CHCHS), 4.14 (1H, dd, J=9.7 Hz,J=4.8 Hz, H—CHCHS), 4.01 (1H, dd, J=11.2 Hz, J=3.0 Hz, H—CHCHONO₂),4.01-3.81 (2H, cs, H—CHCHS, H—CHCHONO₂), 3.30-3.24 (1H, m, CHS), 2.15(3H, s, CH₃).

¹³C-NMR (50 MHz, CDCl₃): 98.65 (CHCHS), 81.55 (CHCHONO₂), 81.26(CHONO₂), 73.87 (CH₂CHS), 69.10 (CH₂CHONO₂) 50.74 (CHS), 14.74 (CH₃).

Example 4 Method to obtain 2-[(R)-methylsulfinyl]isosorbide5-mononitrate (4) and 2-[(S)-methylsulfinyl]isosorbide 5-mononitrate(4bis)

In a 500 mL flask, it was dissolved 7.3 g (32.9 mmol) of2-methylthioisosorbide 5-mononitrate (3) obtained according to Example 3in 75 mL of dioxane. A solution of 7.04 g (32.9 mmol) of NaIO₄ in 110 mLof water was added very slowly, drop by drop, and then stirred for 1hour at room temperature. The mixture was filtered and then the dioxanewas eliminated from the filtrate at reduced pressure. 150 mL of waterwere added. The mixture was extracted with 2×300 mL portions ofchloroform. it was dried over anhydrous magnesium sulfate, filtered andthe solvent eliminated at reduced pressure. This results in 6.6 g of areaction crude containing a mixture of diastereoisomers in a proportionof 65% of (4) and 35% of (4bis). The resulting reaction crude wasrecrystallized from dioxane twice to obtain 2.9 g of the product ofinterest (4) with a purity of 95% by HPLC. Using the mother liquors fromthe first recrystallization, the solvent was eliminated at reducedpressure and the resulting residue recrystallized from dioxane to obtain1 g of the product of interest (4bis) with a purity of 95% by HPLC.

2-[(R)-methylsulfinyl]isosorbide 5-mononitrate (4)

¹H-NMR (200 MHz, CDCl₃): 5.39-5.28 (1H, m, CHONO₂), 5.02 (1H, dd, J=5.6Hz, J=1.6 Hz, CHCHS), 4.89 (1H, t, J=5.5 Hz, CHCHONO₂), 4.29 (1H, dd,J=10.4 Hz, J=6.4 Hz, H—CHCHS), 4.20-3.91 (3H, cs, H—CHCHS, CH₂CHONO₂),3.38-3.31 (1H, m, CHSO), 2.61 (3H, 8, CH₃).

¹³C-NMR (50 MHz, CDCl₃): 82.11 (CHCHONO₂), 81.51 (CHCHS), 80.55(CHONO₂), 69.65 (CH₂CHS) 69.28 (CH₂CHONO₂), 66.24 (CHSO), 37.28 (CH₃).

Microanalysis

calculated (%): 35.44, C; 4.67, H; 5.90, N; 13.51, S; 40.47, O.experimental (%): 35.31, C; 4.67, H; 5.98, N; 13.5, S; 40.60, O.

Mas Spectrometry (LC-(ApcI)MS to 20V): 238 (M+1)⁺

Melting Point: 153° C. by DSC

Monocrystal X-Ray Diffraction: The configuration of the diasteromer (4)is establish as: (R)—S, (S)—C2, (S)—C3, (S)—C4, (R)—C5

2-[(S)-methylsulfinyl]isosorbide 5-mononitrate (4bis)

¹H-NMR (200 MHz, CDCl₃): 5.39-5.28 (1H, m, CHONO₂), 4.89 (1H, t, J=5.6Hz, CHCHONO₂), 4.68 (1H, d, 5.4 Hz, CHCHS), 4.40-3.88 (4H, cs, CH₂CHS,CH₂CHONO₂), 3.48-3.40 (1H, m, CHSO), 2.58 (3H, s, CH₃).

¹³C-NMR (50 MHz, CDCl₃): 82.89 (CHCHS), 82.26 (CHCHONO₂), 80.55(CHONO₂), 69.19 (CH₂CHONO₂), 67.88 (CH₂CHS), 66.94 (CHSO), 36.64 (CH₃).

Microanalysis

calculated (%): 35.44, C; 4.67, H; 5.90, N; 13.51, S; 40.47, O.

experimental (%): 35.65, C; 4.66, H; 5.87, N; 13.56, S; 40.57, O.

Mass Spectrometry (LC-(ApcI)MS to 20V): 238 (M+H)⁺

Melting point: 115° C. by DSC

Monocrystal X-Ray Diffraction: The configuration of the diasteromer(4bis) is establish as: (S)—S, (S)—C2, (S)—C3, (S)—C4, (R)—C5

Example 5 Method to obtain 2-methylsulfonylisosorbide 5-mononitrate (5)

In a flask it was dissolved 1.0 g (4.5 mmol) of 2-methylthioisosorbide5-mononitrate (3) obtained according to example 3 in 20 mL ofacetonitrile. A solution of 4.11 g (18.1 mmol) of periodic acid (H₅IO₆)was added at once, and then stirred during 48 h at room temperature. 50mL of a saturated solution of Na₂SO₃ are added. It is extracted with2×30 mL of methylene chloride. The organic phases are united and theyare washed with 2×30 mL of a saturated solution of Na₂SO₃. It was driedover anhydrous magnesium sulphate, filtered and the solvent eliminatedat reduced pressure. 662 mg are obtained of the product of interest (5).640 mg of the crude are suspended in 25 mL of hexane, filtered, andwashed with 7.5 ml, of chloroform, obtaining 450 mg of the product (5)with a purity of 99.7% by HPLC.

¹H-NMR (200 MHz, DMSO-d₆) 5.53-5.47 (1H, m, CHONO₂), 5.00-4.88 (2H, cs,CHCHONO₂, CHCHS), 4.38 (1H, dd, J=9.8 Hz, J=1.8 Hz, H—CHCHS), 4.12-3.86(4H, cs, H—CHCHS, CH₂CHONO₂, CHSO₂), 3.07 (3H, s, CH₃).

¹³C-NMR (50 MHz, DMSO-d₆): 82.77 (CHCHS), 82.36 (CHCHONO₂), 81.81(CHONO₂), 68.95 (CH₂CHONO₂), 68.46 (CH₂CHS), 67.48 (CHSO₂), 39.31 (CH₃).

Mioroanalysis

calculated (%): 33.20, C; 4.38, H; 5.53, N; 12.66, S; 44.23, O.

experimental (%): 33.45, C; 4.34, H; 5.52, N; 12.69, S; 44.43, O.

Mass Spectrometry (LC-(ApcI)MS to 20V): 254 (M+H)⁺

Melting Point: 173° C. by DSC

Example 6 Method to obtain S-nitroso-2-thioisosorbide 5-mononitrate (6)

In an amber vial, 0.5 g (2.41 mmol) of 2-thioisosorbide 5-mononitrate(1) obtained according to Example 1 were dissolved in 4.0 mL of MeOH,covered and stirred in an ice bath. 320 μL (0.249 g, 2.41 mmol) oftert-butoxynitrite were added and stirred while covered and under coldconditions for 7 hours. The white solid was filtered off and thefiltrate was concentrated to dryness at reduced pressure protected fromexposure to light and at room temperature. This results in 0.48 g(yield: 84%) of a deep red solid identified asS-nitroso-2-thioisosorbide 5-mononitrate (6).

¹H-NMR (CDCl₃, 200 MHz): 5.40-5.25 (so, 1H, CHONO₂), 4.84-4.64 (sc, 2H,CHSNO, CHCHONO₂), 4.40-4.30 (m, 2H, H—CHCHSNO, CHCHSNO), 4.12 (dd, 1H,J=11.4 Hz, J=2.6 Hz, H—CHCHONO₂), 4.00-3.80 (sc, 2H, H—CHCHSNO,H—CHCHONO₂).

¹³C-NMR (CDCl₃, 50 MHz): 88.2 (CHCHSNO), 81.9+81.1 (CHCHONO₂, CHONO₂),73.4 (CH₂CHSNO), 69.5 (CH₂CHONO₂), 51.6 (CHSNO).

Example 7 Method to obtain 2-(tetrahydropyran-2-yl-thio)isosorbide5-mononitrate (7)

0.5 g (2.41 mmol) of 2-thioisosorbide 5-mononitrate (1) obtainedaccording to Example 1 were suspended in 6 mL of 3,4-dihydro-2H-pyranand the mixture cooled in an ice bath. 0.10 g (0.40 mmol) of pyridiniump-toluenesulfonate were added and stirred overnight under an nitrogenatmosphere at room temperature. 50 mL of EtOEt were added and themixture washed with 2×40 mL of a saturated NaCl solution. This resultsin 1.15 g of a reaction crude that is purified by chromatography(Hx:AcOEt=3:2), which yields 0.65 g of the product of interest with apurity of 83%. Recrystallization from hexane gave 0.45 g of the productwith a purity of 95%, identified by its spectroscopy data as2-(tetrahydropyran-2-yl)-2-thioisosorbide 5-mononitrate (7)

¹H-NMR (CDCl₃, 200 MHz): 5.28 (ddd, 1H, J=11.0 Hz, 5.4 Hz, 2.8 Hz,CHONO₂), 5.0-4.85 (sc, 2H, CHSTHP, CHCHONO₂), 4.59 (dd, 1H, J=11.2 Hz,4.6 Hz, CHCHSTHP), 4.20-3.80 (sc, 5H, CH₂CHSTHP, H—CHCHONO₂,CH_(2 THP)), 3.60-3.40 (sc, 2H, H—CHCHONO₂, CH_(THP)), 2.00-1.45 (sc,6H, 3CH_(2 THP)).

¹³C-NMR (CDCl₃, 50 MHz): 89.5 and 89.1 (CHCHS), 83.0 and 81.6(CHCHONO₂), 81.4 and 81.3 (CHSTHP, CHONO₂), 74.9 and 74.1 (CH_(2 THP)),69.0 and 68.9 (CH₂CHSTHP), 64.8 and 64.7 (CH₂CHONO₂), 49.1 and 47.6(CH_(2 THP)) 31.3 and 31.2 (CH_(2 THP)), 25.4 (CH_(2 THP)), 21.7 and21.6 (CH_(2 THP)).

Example 8 Method to obtain 2-(isosorbidyl-2′-dithio)isosorbide5-mononitrate (8)

A solution of 0.170 g (1.72 mmol) of succinimide in EtOH was added to0.323 g (1.56 mol) of 2-thioisosorbide 5-mononitrate (1). After a whiteprecipitate appears, 0.168 mg (2 mmol) of NaHCO₃ were added. Afterstirring at room temperature for 3 hours and 45 minutes an additional100 mg (1.19 mmol) of NaHCO₃ and 10 drops of water were added. After anadditional 1 hour and 30 minutes of stirring, the mixture was brought toreflux. After two hours at reflux, the EtOH was eliminated at reducedpressure, 150 mL of water and 150 mL of AcOEt were added. An emulsion isformed and NaCl is added until the two phases are separated. The organicphase is separated and the aqueous phase washed with 2×150 mL portionsof AcOEt. Each of the three organic phases is washed separately with 100mL of water and the organic phases are combined and dried over anhydrousNa₂SO₄. This is filtered, washed with AcOEt and the solvent eliminatedfrom the filtrate at reduced pressure, obtaining 336 mg of a reactioncrude on which flash chromatography is performed. Use of a mixture of1:1 CHCl₃/AcOEt as an eluent for the chromatographic separation resultsin a fraction of 98 mg of the product of interest (8).

¹H-NMR (300 MHz, CDCl₃): 5.40-5.32 (1H, ca, CHONO₂), 5.04-4.98 (1H, cs,CHCHONO₂), 4.70-4.60 (3H, cs, 3CH), 4.36-4.26 (1H, cs, CHOH), 4.22-4.12(2H, cs, 2H—CH), 4.10-4.00 (3H, cs, 3H—CH), 3.94-3.86 (2H, cs, 2H—CH),3.68-3.56 (3H, cs, 2CH—S, 1H—CH), 2.55 (1H, d, 1H d, J=6.9 Hz, OH).

Infrared spectroscopy (in KBr pellet): 3461 cm⁻¹, 2987 cm⁻¹, 1642 cm⁻¹,1465 cm⁻¹, 1279 cm⁻¹, 1077 cm⁻¹, 846 cm⁻¹.

Microanalysis:

experimental (%): 39.53, C; 34.70, H; 4.77, O; 3.96, N; 17.04 S.

calculated (%): 39.23, C; 34.84, H; 4.66, O; 3.81, N; 17.45, S.

Mass spectrometry:

Electrospray: 368 (M+1).

Electron impact (m/z, (% relative abundance)): 367 (7.4) (M⁺), 261(3.8), 160 (8.6), 129 (15.5), 127 (14.2), 85 (35.7), 69 (100).

Example 9 Step 1 Method to obtain 2-thioisosorbide (9) and2,2′-dithiodiisosorbide (10)

In a 500 mL flask, 15 g (74 mmol) of 2-acetylthioisosorbide, (13)obtained according to WO 00/20420 are dissolved in 225 mL of EtOH. A 85%solution of 11.3 g of KOH in 150 mL of water is added. The resultingmixture is stirred at room temperature for 2 hours, neutralized withAcOH_((e)) and the EtOH eliminated at reduced pressure. The resultingsolution is brought to basic pH by adding NaOH_((a)), and stirred atroom temperature while bubbling an air stream through the solution for10 hours. The solution is acidified with HCl_((o)) and brought to pH=4.The water is eliminated at reduced pressure and the resulting residueredissolved in CH₂Cl₂, filtered and dried over anhydrous Na₂SO₄. Thesolution is filtered, washed and the solvent eliminated from thefiltrate at reduced pressure, obtaining 9.22 g of a reaction crude whichis purified by flash chromatography using different mixtures ofcyclohexane/ethyl acetate as eluent. The elution is started with 3 L ofa 1:1 mixture, and after that, the percentage of the polar solventincreased, first with 2 L of a 3:5 mixture, later with 2 L of a 1:2mixture and finally eluted only with AcOEt. A 2.64 g fraction of thethiol (9) and another 3.06 g of the disulfide (10) are isolated from theeluate.

2-Thioisosorbide (9)

¹H-NMR (200 MHz, CDCl₃): 4.68-4.58 (1H, cs, CHCHOH), 4.48-4.40 (1H, cs,CHCHSH), 4.34-4.18 (1H, bs, s.a. D₂O, CHOH), 4.16-4.06 (1H, m H—CHCHSH),3.96-3.78 (2H, cs, H—CHCHOH, H—CHCHSH), 3.62-3.50 (1H, cs, 1H—CHCHOH),3.48-3.36 (1H, cs, s.a, D₂O, CHS), 2.80-2.60 (1H, bs, d.a. D₂O, OH),1.75 (1H, d, J=8 Hz, d.a. D₂O, SH).

¹³C-NMR (50 MHz, CDCl₃): 90.42 (CHCHSH), 81.47 (CHCHOH), 76.27(CH₂CHSH), 74.00 (CH₂CHOH), 72.04 (CHOH), 43.81 (CHSH).

2,2′-Dithiodiisosorbide (10)

¹H-NMR (200 MHz, CDCl₃): 4.68-4.56 (4H, cs, 2CHCHS, 2CHCHOH), 4.34-4.19(2H, cs, s.a. D₂O, 2 CHOH), 4.19-3.97 (4H, as, 2CH₂CHS), 3.92-3.80 (2H,cs, 2H—CHCHOH), 3.66-3.52 (4H, as, 2H—CHCHOH, 2CHS), 2.63 (2H, d, J=6.6Hz, d.a. D₂O, 20H).

¹³C-NMR (50 MHz, CDCl₃): 87.42 (2CHCHS), 81.98 (2CHCHOH), 73.93(2CH₂CHOH) 72.89 (2CH₂CHS), 72.07 (2CHOH), 54.74 (2CHS).

Step 2 Method to obtain 5,5′-diacetyloxy-2,2′-dithiodiisosorbide (14)and 2-(5′-acetyloxyisosorbidyl-2′-dithio)isosorbide 5-mononitrate (11)

A nitrating mixture is prepared by adding, slowly and with caution at 0°C., 1.8 mL of 60% HNO₃ to a mixture of 7.5 mL of acetic anhydride and12.5 mL of acetic acid. In a 100 flask fitted with reflux cooler,thermometer and magnetic stirrer, 2.77 g (8.6 mmol) of2,2′-dithio-diisosorbide (10) obtained according to Example 8 aredissolved in 17 ml, of acetic acid and 3.5 mL of acetic anhydride areadded. The mixture is cooled at 0° C. in an ice/salt bath. 4 mL of thepreviously prepared nitrating mixture is added dropwise over 15 minutes.It is stirred at 0° C. for 2 hours, observing the solidification of thereaction crude. Then it is stirred for 2 hours and 30 minutes at roomtemperature. The reaction crude is poured over 100 mL of water, filteredand washed with plenty of water. The resulting solid is dried at reducedpressure in the presence of P₂O₅. This results in 2.69 g of a reactioncrude which is purified by preparative reversed phase chromatography. A1:1-mixture of acetonitrile:water is used as an eluent for thechromatography. A 1.01 g fraction of the diacetate (14) (R=COCH₃) andanother 0.5 g fraction of acetate-nitrate (11) (R=NO₂) is isolated.

5,5′-diacetyloxy-2,2′-dithiodiisosorbide (R=COCH₃) (14)

¹H-NMR (200 MHz, CDCl₃): 5.16-5.02 (2H, cs, 2CHOCO), 4.85-4.76 (2H, as,2CH—O—C), 4.63-4.56 (2H, cs, 2CHOC), 4.17-4.05 (2H, cs, 2H—CH),4.00-3.74 (6H, cs, 6H—CH), 3.56-3.48 (2H, cs, 2CHS), 2.09 (6H, s, CH₃).

¹³C-NMR (50 MHz, CDCl₃): 170.27 (2CO), 87.73 (2CHCHS), 80.76 (2CHCHO),73.79 (2CHO), 72.66 (2CH₂CHS), 70.46 (2CH₂CHO), 54.42 (2CHS), 20.63(2CH₃).

2-(5′-Acetyloxyisosorbidyl-2′-dithio)-isosorbide 5-mononitrate (R=NO₂)(11)

¹H-NMR (200 MHz, CDCl₃): 5.37-5.28 (1H, cs, CHONO₂), 5.18-5.06 (1H, cs,CHOCO), 5.02-4.94 (1H, as, CHOC), 4.87-4.78 (1H, cs, CHOC), 4.64-4.56(2H, cs, 2 CHOC), 4.18-3.75 (8H, cs, 4CH₂), 3.59-3.50 (2H, cs, 2 CHS),2.10 (3H, s, CH₃).

¹³C-NMR (50 MHz, CDCl₃): 170.29 (CO), 88.25 (CH), 87.43 (CH), 81.58(CH), 81.16 (CH), 80.79 (CH), 73.80 (CH), 72.78 (CH₂), 72.66 (CH₂),70.51 (CH₂), 69.32 (CH₂), 54.42 (CHS), 53.72 (CHS), 20.65 (CH₃).

Mass spectrometry:

Chemical ionization (NH₃): 410 (M+1)⁺, 427 (M+18)⁺.

Tests for Vasorelaxation

The method used in the assays is substantially the same as described infollowing references:

-   Furchgot, R. F., “Methods in nitric oxide research”; Feelisch &    Stamler, eds., John Wiley & Sons, Chichester, England, pp 567-581.-   Trongvanbichnam K., et al., Jpn. J. Pharmacol. 71 (1996); 167-173.-   Sales, E., at al., Eur. J. Pharmacol. 258 (1994); 47-55.

The different compounds are tested at 5 different concentrations, at aconcentration range from 0.0001 to 10 mM, using from 6 to 9 arterialrings for each compound. The obtained results are compared to those fromthe isosorbide 5-mononitrate, which is used as reference product.

The results are shown in table 1 below and are provided as EC₅₀(effective concentration 50), which is the concentration of each of thetested, compounds yielding a vasodilatation equal to 50% of the maximumtone at which the artery ring has been contracted with 1 μM ofPhenylephrine.

TABLE 1 Test of vasorelaxation Compound EC₅₀ mM (mean ± s.d.) Isosorbide5-mononitrate 0.92 ± 0.2  (1) 0.041 ± 0.006 (2) 0.0053 ± 0.001  (3)0.043 ± 0.006 (4) 0.338 ± 0.01  (6) 0.0012 ± 0.0001 (7)  0.05 ± 0.009(9) 0.024 ± 0.005 (12)  0.023 ± 0.010

As it can be seen from the table, all the compounds tested are morepotent as vasorelaxants than the reference product.

In Vitro Test of the Inhibition of the Platelet Aggregation

The method used in the assays is substantially the same as described infollowing reference:

-   Sales, E., at al., Br. J. Pharmacol. 112 (1994); 1071-1076.

The compounds are tested at four different concentrations, usingplatelet rich plasma from not less than 6 different healthy humandonors. The results obtained are compared to those from 5-isosorbidemononitrate, which is used as reference product.

The results are shown in table 2 and are expressed as IC₅₀ (inhibitoryconcentration 50), which is the concentration of each of the testedcompounds yielding an inhibition equal to 50% of the aggregationobtained with a submaximal concentration (1-4 μM) of ADP (a submaximalconcentration of ADP is the minimal amount of ADP which produces themaximal aggregation).

TABLE 2 Test of inhibition of the aggregation of the platelets (HumanPlatelet Rich Plasma) Compound IC₅₀ mM (mean ± s.d.) sosorbide5-mononitrate 2.68 ± 0.33  (1) 0.11 ± 0.01  (2)  0.01 ± 0.007  (3) 0.41± 0.09  (4) 1.00 ± 0.01  (4bis) 0.85 ± 0.15  (6) 0.0019 ± 0.0005  (7) 0.58 ± 0.021 (12) 0.089 ± 0.01 

As can be seen from table 2, all the compound tested have a potentinhibitory activity on aggregation of the platelets, that is superior tothat of the reference compound.

Inhibition of Human Monocyte and Platelet Adhesion to Human EndothelialCells

The methods used in the assays to determine the effect of the compoundson platelet and monocyte adhesion to human endothelial cells aresubstantially the same as described in the following references:

-   Del Maschio A. et al., J Cell Biol 1996; 135: 497-510-   Bombeli T. et al., Blood 1999; 93: 3831-3838-   Colomé C. et al; Atherosclerosis 2000; 149: 295-302; and-   Martin-Satue M. at al; British Journal of Cancer 1999; 80; 1169-1174

The adhesion of the U937-monocyte cells to confluent humanmicrovasculature endothelial cells (HMEC-1) activated by means of TNF-α(50 ng/ml) and the platelets to human umbilical endothelial cells(HUVEC) were the methods used to determine the inhibitory effect ofcompounds on cellular adhesion. The adhesion of monocytes previouslylabeled with calcein-2M (Molecular Probes) to HMEC-1 activated by TNF-α,was assessed by determination of the associated-cell fluorescence. Theadhesion of washed human platelets previously labeled with MoAb againstCD36-FITC to activated-HUVEC (50 μg/ml of LDL minimally modified, LDLmm)was determined by Laser Scanning Cytometry (LSC, Olympus) determiningthe associated-cell fluorescence. Results are expressed as thepercentage of inhibition respect to control (adhesion of the cells inthe absence of compounds). Table 3 shows the effect of compounds on U937adhesion to activated-HMEC-1. Table 4 shows the effect of compounds onplatelet adhesion to activated HUVEC.

TABLE 3 Inhibition of U937 monocyte adhesion to activated HMEC-1 humanendothelial cells % of inhibition to control Compound (mean ± s.e.m.)(1) 30 ± 8 (12) 15 ± 2

TABLE 4 Inhibition of platelet adhesion to HUVEC activated by LDLmm (50μg/ml) % of inhibition to control Compound (mean ± s.e.m.) (12) 87.5% ±5.1%

Inhibition of LDL Transcytosis in Human Microvascular Endothelial Cells

The method used in the assays to determine the effect of the compoundson LDL transcytosis in human microvascular endothelial cells issubstantially the same as described in Colomé C. et al; Atherosclerosis2000; 149: 295-302. This test allows a prediction on theanti-atherosclerotic potential of the compounds, since the accumulationof LDL in the vascular wall as a consequence of a vascular endothelialcell active transport is one of the first steps in the development ofatherosclerosis.

The method used in the assays to isolate LDL particles from fresh humanplasma and the labelling with Di1 is substantially the same as describedin Pedreño J. et al., Atherosclerosis 2001; 155: 99-112, and Stephan Z.F. and Yuracheck E. C. J. Lipid Res. 1993; 34:325-330

Activated (100 μM histamine) and non activated HMEC-1 were cultivated inthe upper well of inserted of coculture until to reach confluence(Falcon HTS FluorBloK). Then, LPL-Dil (up to 200 μg/ml) was added andcells were incubated for 2 h in the presence and absence of compounds.After these two hours, transcytosis of LDL-Dil particles throughendothelial cells was assessed by determination of the presence offluorescence in the lower well of insert of coculture. Results areexpressed as percentage of inhibition respect to control in the absenceof compounds). Table 5 shows the effect of the compounds on the LDL-Di1transcytosis through HMEC-1.

TABLE 5 Inhibition of LDL transcytosis in human microvascularendothelial cells (% of inhibition to control (mean ± s.e.m.)) CompoundResting cells Stimulated cells (1) 58 ± 15 122 ± 8 (12) 51 ± 19  80 ± 12

Inhibition of LDL Oxidation

The method used in the assays to determine the effect of the compoundson LDL oxidation is substantially the same as described in the followingreferences:

-   Sprenger T., et al., Chem. Phys. Lipids 1998; 91:39-52-   Lynch S. M., et al., Biochim. Biophys. Acta 2000; 1485:11-22-   Pedreño J., et al., Thromb. Res. 2000; 99: 51-60.

The method used in the assays to isolate LDL particles from fresh humanplasma is substantially the same as described in Pedreño J., et al.,Atherosclerosis 2001; 155: 99-112.

Oxidative modification of low-density lipoprotein (LDL) is currentlybelieved to be a central event it the development of atheroscleroticcardiovascular disease. The exposition of LDL to globin-free haemin (thecomplex of ferric ion (Fe³⁺) with protoporphyrin IX), derived from thehaemoglobin in circulating erythrocytes, serves as a physiologicalsource of pro-oxidant Fe³⁺ capable to promoting LDL oxidation. Thecompounds were added in vitro to determine their inhibitory activity onLDL oxidation induced by haemin and H₂0₂. LDL at a final proteinconcentration of 0.2 mg/ml was incubated in the presence of 2.5 μMhaemin and 5 μM H₂0₂. Oxidative modification of LDL particles wasassessed by measurement of conjugated dienes. Experimental samples wereincubated at 37° C. and the increase in absorbance at 234 nm wasautomatically recorded every 5 min for at least 5 h. The effect ofcompounds on the LDL oxidation induced by haemin was tested at 7different concentrations using LDL preparations from 7 different healthydonors. Table 6 shows the inhibitory activity of the compounds at 10 μMexpressed as the percentage of increase of lag phase (time that isrequired so that the reaction of formation of conjugate dienos begins)with respect to control.

TABLE 6 Inhibition of LDL oxidation % of increment of lag phase withrespect to control (mean ± Compound s.e.m.) Isosorbide 5-mononitrate 0 (1) 290 ± 8  (12) 117 ± 56

Inhibition of Plasma Oxidation

The method used in the assays to determine the effect of compounds onthe capacity of oxidation of the plasma is substantially the same asdescribed in Spranger T. et al., Chem Phys Lipids 1998; 91: 39-52.

Lipoprotein oxidation induced in vitro in plasma is expected torepresent a relevant model of the lipoprotein oxidation in the arterialwall. Oxidation of plasma lipoproteins was assessed as the capacity ofoxidation of heparinized plasma and was measured by spectrophotometry asin increase in absorbance at 234 nm. The compounds were added in vitroto determine their inhibitory activity over the capacity of oxidation ofplasma induced by Cu²⁺ (CuSO₄). Heparinized plasma was diluted withphosphate-buffered saline solution (PBS) containing 0.16 M NaCl, and theoxidation was started by 50 μM CuSO₄. Experimental samples wereincubated at 37° C. and the increase in absorbance at 234 nm wasautomatically recorded every 15 min for at least 12 h. The effect ofcompounds over the capacity of oxidation of plasma induced by Cu²⁺ wastested at 7 different concentrations using heparinizaded plasma from 7different healthy donors. Table 7 shows the inhibitory activity of thecompounds at 10 μM expressed as the percentage of increase of lag phasewith respect to control.

TABLE 7 Inhibition of plasma oxidation % of increment of lag phase withrespect to control (mean ± Compound s.e.m.) (1) 245 ± 26 (2)  7 ± 1(12)  200 ± 10 Isosorbide 5-mononitrate 0

Preventive and Curative Effect on Atherogenesis in Rabbits Fed on HighCholesterol Diet

The method used is substantially the same as described in Shore B, ShoreV., In: Day CE (ed) Atherosclerosis Drug Discovery, Plenum Press, NewYork and London, pp 123-141, 1976.

Twenty-one male New Zealand White rabbits (10 weeks old at the beginningof the protocol) were maintained under standardized conditions (22° C.,40% to 60% humidity) with regular day/night cycle and free access towater. The animals were randomly assigned to 1 of 3 groups. Group 1(n=5) received standard maintenance diet for 75 days; group 2 (n=16)received the same diet but supplemented with 1% (wt/wt) cholesterol for45 days. At the end of this period, group 2 was classified into tworandomly groups. The treated-group (n=9) receiving for additional 30days 1.9 mg/kg/day of compound 12 (maintaining the 1% cholesterol in itsdiet), and the non-treated group (n=17) receiving a diet with a 1% ofcholesterol for additional 30 days.

Study variables included triglycerides and total cholesterol. Animalswere euthanized on day 75 after the start of the protocol withpentobarbital i.v. The entire aorta was removed 1 or 2 cm into the iliacarteries and fixed in buffer phosphate 0.1 M (pH 7.4) withglutaraldehyde (4%). Macroscopic and microscopic analyses of the sampleswere performed in a blinded fashion. Aortas were dissected and stainedwith Red Oil. Adventitial fat was removed, and aortas were openedlongitudinally, immersed in Red Oil solution for overnight, washed inpropylenglycol and fixed onto a flat surface. Images of the aortas weretaken with a standard camera. Subsequently, 3 sections were taken fromthe aortic arch area, the thoracic aorta, and at the abdominal aorta(renal orifices). Sections were paraffin-embedded, were cut with amicrotome Ultracut (Spain) and stained conventionally with hematosilineeosin. Images of aortas stained with Red Oil and hematosiline eosin weretaken with a standard camera. Images were imported into Photoshop(Adobe) with a AGFA slide scanner. By means of the procedure previouslydescribed by Lillie RD (Lillie, R. D., 1994, Stain Technology, vol. 19,pp 55) the surface of injury in all the aorta and the thickness of theintima in histological samples was determined. The surface or area ofthe atherosclerotic injury (evaluated from the stained aortas with theRed Oil) are determined by the number of pixels of the image turned tomm². The intima thickness of the arteria (evaluated from the stainedhistological samples with Haematosiline-Eosin) was quantified in mm in asimilar way to that described the surface or area of the atheroscleroticinjury. For it, the sections that contained injuries of fatty striaewere selected and the thickness of the intima was determined (from thearterial media to endothelium). The mean thickness of the lesion areawas assessed in representative sections per aortic quadrant, and thestatistical mean was calculated. The fasting plasmatic totalcholesterol, HDL cholesterol and triglycerides levels are shown in table8. The statistical examination (Student t) has revealed no significantdifference between the medicated group and the control group (the groupnot medicated but with high cholesterol diet) in regard to plasma lipid.

TABLE 8 Plasmatic lipid profile [mg/dl] (mean ± s.e.m.) Treated with thecompound Control example (12) Basal End Basal End Total 42 ± 4.4 1073.9± 4.9 33.3 ± 4.1 1461 ± 147 cholesterol Triglycerides 185 ± 50     312.7± 130 121 ± 34 347 ± 82

The area rate covered by the atherosclerotic lesions are shown in table9.

TABLE 9 Atherosclerotic lesions Area percent of atherosclerotic lesions(%) Group (mean ± s.e.m.) Control 62 ± 6 Treated with the compound 15 ±3 example (12)

The thickness of intimal layer of the aorta is shown in table 10.

TABLE 10 Thickness of the intimal layer of the aorta Group Thickness mm(mean ± s.e.m.) Control 0.323 ± 0.013 Treated with the compound 0.096 ±0.009 example (12)

In table 8 it is shown that there was no difference in plasma lipidsbetween the control and the medicated group thereby demonstrating thatthe compound (12) did not influence upon lipid metabolism. As seen intable 9 and in table 10, both area percent of the atheroscleroticlesions as the intimal thickness were significantly reduced in the grouptreated with the compound (12) in comparison with the control group.

Preventive and Curative Effect on Atherogenesis in Apo E-Deficient Mouse

The preventive and therapeutic effect of the present compounds onatherosclerosis will be also illustrated by way of the followingexample.

Preventing and curative effect on atherogenesis of the compounds it isinvestigated in apo E-deficient mouse fed on standard diet model. Maleand female apo E-deficient mouse (OLA129X C57/BL6J) of three months oldwere fed on standard diet. The control group included 8 males and 7females mouse, while the treated group consist of 8 males and 7 femalesmouse. All the animals had similar cholesterol and body weight at thebeginning of the experiment. Both the control and the treated groupswere studied for 12 weeks. The treated group received 5 mg/Kg/day of thecompound (12).

Plasma lipid parameters (total cholesterol, HDL cholesterol andtriglycerides) and oxidative stress (measured as 8-iso-prostaglandin F2αlevels) were measured at predetermined intervals. After completion ofthe administration period, the thoracic aorta was isolated and stainedto determine the area of the atherosclerotic injuries deposited in theinternal wall of the blood vessel in according with the Red Oil method(Lillie, R. D., 1994, Stain Technology, vol. 19, pp 55).

The fasting plasmatic levels of total cholesterol, HDL cholesterol,triglycerides and 8-iso-prostaglandin F2α levels are shown in table 11.

TABLE 11 Plasmatic lipids and oxidative stress parameters (mean ± s.d.)Male Female Treated Treated with with the compound compound Control (12)Control (12) Total cholesterol 14.3 ± 2   18 ± 3  9.5 ± 2   11.3 ± 1.5 (mmol · L-1) HDL-cholesterol  0.5 ± 0.15 0.47 ± 0.15 0.4 ± 0.1 0.48 ±0.13 (mmol · L-1) Triglycerides 2.6 ± 0.6 1.5 ± 0.4 1.9 ± 0.6 1.3 ± 0.2(mmol · L-1) 8-iso- 273 ± 19  101 ± 13  182 ± 19  99 ± 7  prostaglandinF2α (pg · mL-1)

The area rate covered by the atherosclerotic lesions on the inner wallof the blood vessel and the area covered with macrophages are shown intable 12.

TABLE 12 Area of Area covered with atherosclerotic macrophages [μm²]lesions [μm²] (mean ± Group (mean ± s.e.m.) s.e.m.) Control 80 ± 27 60 ±8 Treated with 55 ± 19 45 ± 9 the compound example (12)

In table 11 it is shown that compound (12) is able to decrease thelevels of triglycerides in plasma as well as the oxidative stress ofthose animals. In table 12 it is shown how the compound (12) reducesboth the area of the atherosclerotic lesions and the area covered withmacrophages.

In Vivo Antithrombotic Effect

The method used is substantially the same as described by Kurz (Kurz K.D., et al., Thromb. Res. 1990, 60:269-280) and modified by Feuerstein(Feuerstein G. Z., et al., Artherioscler. Thromb. Vase. Biol. 1999, 19:2554-25562).

Rats received a single oral dose of 100 mg/Kg of the compounds.

Forty-five minutes after dosing rats were anaesthetized with sodiumpentobarbital (40 mg/kg, i.p.) and they were placed later dorsally on aheated (37° C.) surgical board.

The left carotid artery was isolated and a Parafilm 4 sheet (7×20 mm,American National Can) was placed under it. An electromagnetic soundingof flow (Transonic Systems Inc.) was placed on the artery to measureblood flow.

Sixty minutes after product administration, a paper patch saturated withFeCl₃ solution (70%) was placed (and not removed for the whole durationof the experiment) on the left carotid artery downstream from thesounding of flow to initiate thrombosis. The blood flow was controlledduring the 60 min later to the application of the patch in the artery.

The vessel was considered totally occluded by the thrombus formed whenno blood flow was detected (0.0 ml/min). In this model, thrombusformation usually takes place within 15 to 20 minutes in non-treatedanimals. An animal was considered as fully protected by treatment if athrombus did not occlude the vessel during the period of study (60 minafter FeCl₃ continue patch application).

The results are shown in table 13 and are expressed as percentage ofanimals fully protected by the treatment.

TABLE 13 In vivo anti-thrombotic activity Compound % of animals fullyprotected 1 100 3 100 4 80 12 71

As it can be observed in table 13, all the compound tested have a potentin vivo anti-thrombotic activity.

In Vivo Synergistic Anithrombotic Effect

The method used is substantially the same as described by Kurz (Kurz K.D., at al., Thromb. Res. 1990, 60: 269-280) and modified by Feuerstein(Feuerstein G. Z., et al., Artherioscler, Thromb. Vasc. Biol. 1999, 19:2554-25562).

Rats received a single oral dose of one or the combination of twocompounds as described in table 14. The doses of the compounds used didnot modify the blood pressure nor the heart rate of the animals.

Forty-five minutes after dosing administration, rats were anaesthetizedwith sodium pentobarbital (40 mg/kg, i.p.) and they were placed laterdorsally on a heated (37° C.) surgical board.

The left carotid artery was isolated and a Parafilm M sheet (7×20 mm,American National Can) was placed under it. An electromagnetic soundingof flow (Transonic Systems Inc.) was placed on the artery to measureblood flow.

Sixty minutes after product administration, a paper patch saturated withFeCl₃ solution (70%) was placed (and not removed for the whole durationof the experiment) on the left carotid artery downstream from thesounding of flow to initiate thrombosis. The blood flow was controlledduring the 60 min later to the application of the patch in the artery.

The vessel was considered totally occluded by the thrombus formed whenno blood flow was detected (0.0 ml/min). In this model, occlusivethrombus formation usually takes place within 15 to 20 minutes innon-treated animals. An animal was considered as fully protected bytreatment if a thrombus did not occlude the vessel during the period ofstudy (60 min after FeCl₃ continue patch application).

The fractional product concept was used to identify synergy between thecompounds. In accordance with this concept, if we consider the fullprotection of the animal as:

${{Fractional}\mspace{14mu} {inhibition}} = {( {1 - \frac{A}{B}} ) \times 100}$

where A is the rate of animals with thrombotic occlusion in treatedgroup; and B is the rate of animals with thrombotic occlusion in thecontrol group.

For two drugs that act of independent way:

${{Fractional}\mspace{14mu} {inhibition}} = {\lbrack {1 - ( {\frac{A_{1}}{B} \times \frac{A_{2}}{B}} )} \rbrack \times 100}$

where A₁ is the rate of animals with thrombotic occlusion in thetreatment group A₁; A₂ is the rate of animals with thrombotic occlusionin the treatment group A₂; and B is the rate of animals with thromboticocclusion in the control group

If the protection obtained with the combination of the drugs is higherthan the fractional inhibition for two compounds acting independentlythen synergism is considered to occur.

The results are shown in table 14 and are expressed as rate of animalswith thrombotic occlusion in each group.

TABLE 14 Rate of animals with thrombotic occlusion (% of Group animalsprotected) Control 1 (0%) Acetyl salicylic acid (100 mg/Kg/ 0.50 (50%)day, oral, for 3 days) Compound 12 (22.3 mg/Kg, oral, 0.5 (50%) acutethe day of study) Acetyl salicylic acid (100 mg/Kg/ 0 (100%) day, oralfor 3 days) + compound 12 (22.3 mg/Kg, oral, acute the day of study)Clopidogrel (1.5 mg/Kg/day, 0.50 (50%) oral for 3 days) Clopidogrel (1.5mg/Kg/day, 0.17 (83%) oral for 3 days) + compound 12 (22.3 mg/Kg, oral,acute the day of study)

${{Fractional}\mspace{14mu} {inhibition}\mspace{14mu} ( {{{acetyl}\mspace{14mu} {salicyclic}\mspace{14mu} {acid}} + {{compound}\mspace{14mu} 12}} )} = {\quad{{\lbrack {1 - ( {\frac{0.5}{1} \times \frac{0.5}{1}} )} \rbrack \times 100} = {75\%}}}$

As the percentage of animals protected with the combination of the twodrugs (acetyl salicylic acid+compound 12) is higher than 75% (it is100%) there is a synergism between the two products.

${{Fractional}\mspace{14mu} {inhibition}\mspace{14mu} ( {{clopidogrel} + {{compound}\mspace{14mu} (12)}} )} = {{\lbrack {1 - ( {\frac{0.5}{1} \times \frac{0.5}{1}} )} \rbrack \times 100} = {75{\%.}}}$

As the percentage of animals protected with the combination of the twodrugs (clopidogrel+compound 12) is higher than 75% (it is 83%) there isa synergism between the two products.

In Vitro Protection Against the Cytotoxicity Induced by Oxygen RadicalsUsing the XTT-Based Method in HUVEC Cells.

The method used is substantially the same as described by Caveda L., etal. (J. Clin. Invest. 1956; 98: 886-893).

For the determination of the capacity to inhibit the cytotoxicityinduced by oxygen radicals Human Umbilical Vein Endothelial Cells(HUVEC) were used. HUVEC were isolated and cultured in M199 and 20% FCS(Fetal Calf Serum).

Test XTT assay is based on hydrolysis by the metabolically active cellsof the salt of tetrazolio XTT to form an orange product, the formazandye formed is soluble and is directly quantified using anspectrophotometer.

HUVEC cells were cultivated until a sub-confluence status in a 96 welltissue culture plate and pre-treated with 50 μM of the compound for onehour. After that, cells were treated with 800 μM of peroxynitrite for anovernight.

After overnight incubation cells were incubated with the yellow XTTsolution (final concentration 0.3 mg/ml) for 4 hours. After thisincubation period, orange formazan solution was formed, which wasspectrophotometrically quantified using an ELISA plate reader at 450 nm

The results are shown in table 15 and are expressed as percentage ofdeath cells.

TABLE 15 In vitro cytoprotective activity Compound % of death cells 1 802 80 3 80 12 50

As it can be observed in table 15, all the compound tested have a cellprotective activity (P<0.05) against the cell damage induced by oxygenradicals.

In Vivo Protection Against Ischemia-Reperfusion Damage in the Heart.

Experiments were performed according to the model previously describedby Hirata Y at al (Journal of Cardiovascular Pharmacology, 1998, 31:322-326)

After a food fasting period of 6 hours, animals were divided into groupsof at least 8 animals each. Doses of products were given by oralsounding 1 h before the ischemia induction. Animals were anaesthetizedwith pentobarbital (40 mg/kg, i.p.) and the standard limb lead IIelectrocardiogram was recorded to detect the S-wave depression. 0.3 U/kgarginine-vasopressin (AVP) (Sigma Chemicals, St Louis, Mo., USA) wasinjected into the carotid artery to induce vasoconstriction of smallcoronary arteries and increase in coronary resistance. In all the groupsreceived AVP 60 min after, the test drug. In all groups, after AVPinjection of 10 minutes ECG recording was performed.

The results are shown in table 16 and are expressed as S-wave decrements(μvolts).

TABLE 16 Protection of S-wave S-wave Compound dose (mg/kg, p.o.)decrement (μV) decrement (%) Vehicle — 46.2 ± 4.9  — 12  20 22.6 ± 5.9 52 12  100 8.6 ± 2.8 81 9 20 16.5 ± 9.8  64 9 100 9.7 ± 4.6 79 4 20 36.5± 11.6 21 4 100 5.9 ± 3.9 87

As it can be observed in table 16, all the compounds tested protectagainst ischemia-reperfusion damage in the heart.

1.-39. (canceled)
 40. A method for the treatment of a condition selectedfrom atherosclerosis, endothelial dysfunctions, vasospasm, cardiacallograft vasculopathy, dysfunctions of the circulatory system, plateletactivation, thrombosis stroke, pathological conditions where oxidationstress causes or results in pathogenesis, pathological condition whereina deficit of nitric oxide causes or results in pathogenesis, and/ortissue damage resulting from ischemia or ischemia neperfusion in apatient, said method comprising administering to said patient in need oftreatment a therapeutically effective amount of at least one compound offormula (I) or a tautomer, a pharmaceutically acceptable salt, a prodrugor a solvate thereof:

wherein: n is an integer of 0, 1, or 2, X represents —S(O)_(m)—, —(C═O)—or a single bond, wherein m is an integer of 0, 1, or 2, with theproviso that when X represents —(C═O)— then n is 0, R representshydrogen or is a residue R^(a), which residue R^(a) is selected from thegroup consisting of: C₁₋₆ alkyl; C₂₋₆ alkenyl; C₃₋₈ cycloalkyl; C₃₋₈cycloalkyl, wherein one CH₂ group is replaced by O, S, NH or NCH₃; C₄₋₈cycloalkenyl; C₄₋₈ cycloalkenyl, wherein one CH₂ group is replaced by O,S, NH or NCH₃; phenyl; pyridyl; thiophenyl; nitrosyl; S-cysteinyl;S-glutathionyl; and

wherein R* is selected from the group consisting of hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₃₋₈ cycloalkyl, C₄₋₈ cycloalkenyl, acetyloxy,hydroxyl, ONO₂ and halogen; wherein R^(a) optionally is substituted byone to three groups independently selected from C₁₋₆ alkyl, C₂₋₆alkenyl, C₃₋₈ cycloalkyl, C₄₋₈ cycloalkenyl, acetyloxy, hydroxyl, ONO₂and halogen.
 41. The method according to claim 40 wherein either one orboth of m and n is
 0. 42. The method according to claim 40 wherein Xrepresents a single bond or —S—.
 43. The method according to claim 40wherein R represents hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₃₋₈cycloalkyl, C₄₋₈ cycloalkenyl, (C₁₋₆ alkyl) C₃₋₈ cycloalkyl, (C₁₋₆alkyl) C₄₋₈ cycloalkenyl, phenyl or (C₁₋₆ alkyl)phenyl.
 44. The methodaccording to claim 40 wherein R is C₁₋₆ alkyl.
 45. The method accordingto claim 40 wherein the compound according to formula (I) is a compoundaccording to formula (Ic) or (Id):


46. The method according to claim 40 wherein the compound of formula (I)is selected from: 2-thioisosorbide 5-mononitrate,5,5′-dinitrate-2,2′-dithiodiisosorbide, 2-methylthioisosorbide5-mononitrate, 2-[(R)-methylsulfinyl]isosorbide 5-mononitrate,2-[(S)-methylsulfinyl]isosorbide 5-mononitrate,2-methylsulfinylisosorbide 5-mononitrate, 2-methylsulfonylisosorbide5-mononitrate, S-nitroso-2-thioisosorbide 5-mononitrate,2-(tetrahydropyran-2-yl-thio)isosorbide 5-mononitrate,2-(isosorbidyl-2′-dithio)isosorbide 5-mononitrate, and2-(5′-acetyloxyisosorbidyl-2′-dithio)isosorbide 5-mononitrate.
 47. Themethod according to claim 40 wherein the compound is2-acetylthioisosorbide 5-mononitrate, which is represented by thefollowing formula:


48. The method according to claim 40 which additionally comprises athrombolytic agent.
 49. The method according to claim 48 wherein thethrombolytic agent is a plasminogen activator.
 50. The method accordingto claim 48 wherein the thrombolytic agent is urokinase, streptokinase,alteplase or anistreplase.
 51. The method according to claim 40 in whichan anticoagulant agent is additionally present.
 52. The method accordingto claim 40 wherein the anticoagulant agent is heparin, dicoumarol,acenocoumarol, enoxaparine or pentosan polysulfate.
 53. The methodaccording to claim 40 in which an antithrombotic agent is additionallypresent.
 54. The method according to claim 53 wherein the antithromboticagents is acetylsalicylic acid, dipyridamole, ticlopidine, clopidrogel,triflusal, pentosan polysulfate or abciximab.
 55. The method accordingto claim 40 in which an immunoglobulin or a fragment thereof having aspecificity for glycoprotein is additionally present.
 56. The methodaccording to claim 40 which additionally comprises an hypolipemiantagent.
 57. The method according to claim 56 wherein the hypolipemiantagents is simvastatin, lovastatin, atorvastatin, pravastatin,fluvastatin, eptastatin, lifibrol, acifran, acitemate, flunicate orrosuvastatin.
 58. The method according to claim 40 in which anantioxidant/free radical scavenger agent is additionally present. 59.The method according to claim 58 wherein the antioxidant/free radicalscavenger agent is nicaraven, ranolazine, emoxipin, glutathione,edaravone, raxofelast, lycopene, N-acetyl-L-cysteine,N-acetyl-D-cysteine, a racemic mixture of N-acetyl-L-cysteine andN-acetyl)-D-cysteine, or carvedilol.
 60. The method according to claim40 wherein the pathological conditions where oxidative stress causes orresults in pathogenesis is selected from allergies, stroke, Alzheimer'sdisease, and ischemic cardiovascular disease.